Dipeptidyl peptidase IV (DP IV) is a serine protease which cleaves N-terminal dipeptides from a peptide chain containing, preferably, a proline residue in the penultimate position. Although the biological role of DP IV in mammalian systems has not been completely established, it is believed to play an important role in neuropeptide metabolism, T-cell activation, attachment of cancer cells to the endothelium and the entry of HIV into lymphoid cells.
Likewise, it has been discovered that DP IV is responsible for inactivating glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide also known as gastric-inhibitory peptide (GIP). Since GLP-1 is a major stimulator of pancreatic insulin secretion and has direct beneficial effects on glucose disposal, in WO 97/40832 and U.S. Pat. No. 6,303,661 inhibition of DP IV and DP IV-like enzyme activity was shown to represent an attractive approach for treating non-insulin-dependent diabetes mellitus (NIDDM).
The present invention provides a new use of DP IV-inhibitors for the treatment of conditions mediated by inhibition of DP IV and DP IV-like enzymes, in particular the treatment of neuronal disorders and immune disorders including multiple sclerosis, and pharmaceutical compositions e.g. useful in inhibiting DP IV and DP IV-like enzymes and a method of inhibiting said enzyme activity.
This invention relates to a method of treatment, in particular to a method for the treatment of central nervous disorders, immune and autoimmune disorders, especially multiple sclerosis and to compositions for use in such method. Dipeptidyl peptidase IV (DP IV) is a post-proline (to a lesser extent post-alanine, post-serine or post-glycine) cleaving serine protease found in various tissues of the body including kidney, liver, and intestine.
It is known that DP IV-Inhibitors may be useful for the treatment of impaired glucose tolerance and diabetes mellitus (International Patent Application, Publication Number WO 99/61431, Pederson R A et al, Diabetes. 1998 August; 47(8):1253-8 and Pauly R P et al, Metabolism 1999 March; 48(3):385-9). In particular WO 99/61431 discloses DP IV-Inhibitors comprising an amino acid residue and a thiazolidine or pyrrolidine group, and salts thereof, especially L-threo-isoleucyl thiazolidine, L-allo-isoleucyl thiazolidine, L-threo-isoleucyl pyrrolidine, L-allo-isoleucyl thiazolidine, L-allo-isoleucyl pyrrolidine, and salts thereof.
The term DP IV-like enzymes relates to structurally and/or functionally DP IV/CD26-related enzyme proteins (Sedo & Malik, Dipeptidyl peptidase IV-like molecules: homologous proteins or homologous activities? Biochimica et Biophysica Acta 2001, 36506: 1-10). In essence, this small group of enzymes has evolved during evolution to release H-Xaa-Pro-Dipeptides and H-Xaa-Ala-Dipeptides from N-terminus of oligo- or polypeptides. They show the common feature, that they accomotate in the Pro-position also Al, Ser, Thr and other amino acids with small hydrophobic side-chains as, Gly or Val. The hydrolytic efficacy is ranked Pro>Ala>>Ser, Thr>>Gly, Val. Same proteins have been only available in such small quantities, that only the post-Pro or post-Ala cleavage could be established. While the proteins: DP IV, DP II, FAPα (Seprase), DP 6, DP 8 and DP 9 are structurally related and show a high sequence homology of, attraction is an extraordinary functional DP IV-like enzyme, characterized by a similar activity and inhibitory pattern.
Dipeptidyl peptidase IV (CD26) has to be shown an activation marker for T, B, and NK cells (Schön, E., Demuth, H. U., Barth, A., and Ansorge, S. (1984) Biochem. J. 223, 255-258, Bühling, F, Junker, U, Reinhold, D, Neubert, K, Jäger, L, and Ansorge, S. Functional role of CD26 on human B lymphocytes. Immunol. Lett. 45, 47-51. 1995; Ansorge, S., Schön, E., and Kunz, D. Membrane-bound peptidases of lymphocytes: functional implications. Biomed. Biochim. Acta 50, 799-807. 1991; Schön, E., Eichmann, E., Grunow, R, Jahn, S., Kiessig, S. T., Volk, H, and Ansorge, S. Dipeptidyl peptidase IV in human T lymphocytes. An approach to the role of membrane peptidases in the immune system. Biomed. Biochim. Acta 45, 1523-1528. 1986.). Surface expression of CD26 is up regulated after mitogenic, anti-CD3 or IL-2 stimulation of T cells, S. aureus protein stimulation of B cells and IL-2 stimulation of NK cells (Reinhold, D, Bank, U., Bühling, F, Träger, M, Born, I., Faust, J., Neubert, K., and Ansorge, S. Inhibitors of dipeptidyl peptidase IV (DP IV, CD26) induces secretion of transforming growth factor-β1 (TGF-β1) in stimulated mouse splenocytes and thymocytes. Immunol. Lett. 58, 29-35. 1997).
Stimulation of human T cells by pokeweed mitogen results in a significant increase of IL-2, IFN-γ, and DP IV mRNA expression. The changes observed in cytokine mRNA expression are dose-dependently suppressed by a specific inhibitor of dipeptidyl peptidase IV (Arndt, M., Lendeckel, U., Spiess, A, Faust, J., Neubert, K., Reinhold, D, and Ansorge, S. Dipeptidyl Peptidase IV (DP IV/CD26) mRNA Expression in PWM-Stimulated T-cells Is Suppressed by SpecificDP IV Inhibition, an Effect Mediated by TGF-β1. Biochem Biophys. Res Commun. 274, 410-414. 2000.). This effect seems to be mediated by TGF-β1.
Multiple sclerosis (MS) is a demyelinating disease of the central nervous system with a presumed autoimmune pathogenesis involving autoantigen-specific CD4+ T cells and cytokines (Rohowsky-Kochan, C, Molinaro, D, and Cook, S D. Cytokine secretion profile of myelin basic protein-specific T cells in multiple sclerosis. Multiple Sclerosis 6, 69-77. 2001.). Myelin basic protein (MBP)-specific, CD4+ T cell clones (TCC) derived from patients with multiple sclerosis express high levels of DP IV/CD26. Specific inhibition of dipeptidyl peptidase IV results in a dose-dependent suppression of DNA synthesis and IFN-γ, IL-4, and TNF-α production of the antigen-stimulated TCC (Reinhold, D, Hemmer, B, and Gran, B. Dipeptidyl peptidase IV (CD26): role in T cell activation and autoimmune disease. Adv. Exp. Med. Biol. 477, 155-160. 2000.).
Inhibitors of dipeptidyl peptidase IV suppress activation of human MBP-specific CD4+ T cell clones (Reinhold, D, Hemmer, B, Gran, B, Born, I., Faust, J., Neubert, K., McFarland, H F, Martin, R, and Ansorge, S. Inhibitors of dipeptidyl peptidase IV/CD26 suppress activation of human MBP-specific CD4+ T cell clones. J Neuroimmun 87(1-2), 203-209. 1998.).
The degeneration underlying MS results from degradation of the myelin sheath, an electrically insulating fatty layer that surrounds nerve fibers and permits the rapid conduction of electrical signals. This loss of myelin can seriously impair the ability of neurons to conduct an electrical signal effectively. Symptoms will depend on where in the central nervous system (CNS) the myelin loss occurs and, thus, which nerve pathways become impaired.
The disease appears to be autoimmune in nature, i.e., the body's own immune system is responsible for the damage. The principal target of the autoimmune reaction appears to be Myelin Basic Protein (MBP), although other MS antigens have been proposed. In the early stages of the disease, a type of CNS cell called an oligodendrocyte can repair this damage and replace the lost myelin. However, these cells can also be destroyed in MS and so sufferers may lose the ability to repair the damage over time, which allows the disease to progress.
The mechanism of disease progression appears complex and several components of the immune system have been linked to the disease. While the underlying tissue damage appears to result predominantly from a T cell mediated response, antibodies against MS antigens are often present in the cerebrospinal fluid (CSF) and at active lesions. Antibodies are not normally present in the CSF and some disruption of the blood brain barrier (BBB), a protective membrane around the CNS, must also occur to allow antibodies, T cells and macrophages to enter the CSF. This change in the permeability of the BBB appears to be one of the defining events in the development of MS.
Activated macrophages secrete pro-inflammatory cytokines such as TNF-α and interferon-γ, leading to the production of destructive enzymes and free radicals. In addition to the complex nature of the immune response itself, the disease can also affect a number of targets. As well as the myelin sheath, other cells such as astrocytes and microglia can be attacked, forming discreet regions of damage known as plaques or lesions. The lesions are also known as scleroses (hence the name) and can occur in both the brain and the spinal cord. The sites of the lesions tend to be near blood vessels and are commonly found on the optic nerve, cerebellum, periventricular regions and spinal cord.
The initial mechanism for the onset of disease remains largely unknown and the number of active lesions at any given time is actually quite low. The heterogeneous nature of the disease and the number of clinical subtypes that this creates suggest that MS is probably a series of related conditions rather than one disease.
DP IV-inhibitors were investigated in the EAE mouse, an animal model for MS (A. Steinbrecher, D. Reinhold, L. Quigley, A. Gado, N. Tresser, L. Izikson, I Born, J. Faust, K. Neubert, R. Martin, S. Ansorge, and S. Brocke. Targeting dipeptidyl peptidase IV (CD26) suppresses autoimmune encephalomyelitis and up-regulates TGF-beta 1 secretion in vivo. J Immunol 166 (3):2041-2048, 2001; A. Steinbrecher, D. Reinhold, L. Quigley, A. Gado, N. Tresser, L. Izikson, I Born, J. Faust, K. Neubert, R. Martin, S. Ansorge, and S. Brocke. Dipeptidyl peptidase IV in inflammatory CNS disease. Adv Exp Med Biol 477:145-153, 2000.)
Inhibitors of dipeptidyl peptidase IV exert anxiolytic-like action when administered centrally (icv) in rats. Chronic treatment with the antidepressant Imipramine has recently been demonstrated to reduce the severity of acute MPB-induce EAE in female Lewis rats (Stephan, M., Straub R. H., Breivik T., Pabst, R. and Hörsten, S. v. Postnatal maternal deprivation aggravates experimental autoimmune encephalomyelitis (EAE) in adult Lewis rats: reversal by chronic imipramine treatment. Int. J. Dev. Neurosci. in press (2002)).
Several studies have underlined the importance of the relationship between stressful life events, psychological distress, and clinical exacerbation of multiple sclerosis (MS) in humans. Stress modulates the course of EAE in rats (Dimitrijevic, M., Laban, O., Hörsten, S. v., Markovic, B. M. & Jankovic, B. D. (1994). Neonatal sound stress and development of experimental allergic encephalomyelitis in Lewis and DA rats. Int J Neurosci, 78, 135-43; Laban, O., Dimitrijevic, M., Hörsten, S. v., Markovic, B. M. & Jankovic, B. D. (1995). Experimental allergic encephalomyelitis in adult DA rats subjected to neonatal handling or gentling. Brain Res, 676, 133-40.). Stress protection is currently not considered as a treatment option for MS.
Further examples of low molecular weight dipeptidyl peptidase IV inhibitors are agents such as tetrahydroisoquinolin-3-carboxamide derivatives, N-substituted 2-cyanopyroles and -pyrrolidines, N-(N′-substituted glycyl)-2-cyanopyrrolidines, N-(substituted glycyl)-thiazolidines, N-(substituted glycyl)-4-cyanothiazolidines, amino-acyl-borono-prolyl-inhibitors and cyclopropyl-fused pyrrolidines. Inhibitors of dipeptidyl peptidase IV are described in U.S. Pat. Nos. 6,011,155; 6,107,317; 6,110,949; 6,124,305; 6,172,081; WO 99/61431, WO 99/67278, WO 99/67279, DE 198 34 591, WO 97/40832, DE 196 16 486 C 2, WO 98/19998, WO 00/07617, WO 99/38501, WO 99/46272, WO 99/38501, WO 01/68603, WO 01/40180, WO 01/81337, WO 01/81304, WO 01/55105, WO 02/02560 and WO 02/14271, the teachings of which are herein incorporated by reference in their entirety.